Particle functionalization and characterization

The detection and study of specific proteins in a heterogeneous solution requires functionalized probe particles that have a strong affinity for the target protein while suppressing non-specific interactions. To this end we functionalize magnetic and plasmonic probe particles through covalent (e.g. gold-thiol, EDC-NHS) and strong non-covalent interactions (e.g. biotin-streptavidin). Non-specific interactions are typically suppressed by bovine serum albumin (BSA) or polyethylene glycol (PEG) coatings. Each analyte requires a tailored functionalization protocol, for which we base ourselves on robust and proven chemical procedures. We characterize our functionalized particles using techniques such as dynamic light scattering, spectroscopy and microscopy. A few examples of recent implementations are given below.

For plasmonic particles the signal that is generated by the presence of the analyte depends on the local field strength. We mostly employ gold nanorods as our workhorse particle [1] , which exhibit a strongly enhanced field around the tips of the particle [2]. To fully exploit this high field at the tips we have developed protocols to specifically functionalize the tips of the particle using thiolated receptors. Tip-specific functionalization is possible with a two-step process. First the gold nanorod is incubated in an aqueous solution of surfactant (blue) that forms a bilayer on the surface of the particle. At the tips, were the curvature is large, the bilayer is less dense leading to an anistropic rate of functionalization due to lower steric hindrance [3].

(a) Numerical calculation of the electromagnetic field strength near a gold nanorod, showing a strongly enhanced field around the tips of the particle. (b) Schematic of the protocol used to selectively functionalize the tips of a gold nanorod with thiolated receptors (brown molecules) by exploiting the steric hindrance provided by a dense bilayer of surfactant (blue molecules) [3].

For superparamagnetic particles we employ various tailored functionalization protocols that exploit different coupling chemistries. Two basic methods are physisorption and covalent coupling by EDC-NHS chemistry. Another approach is to functionalize the particles by covalent as well as strong non-covalent coupling methods. The below figure shows an example where both methods have been used: biotinylated PEG linkers are coupled to antibodies via NHS chemistry, and these are subsequently bound to streptavidin-coated nanoparticles [4]. This molecular architecture has been shown to be functional in undiluted blood plasma.

Scheme of a molecular surface architecture on magnetic nanoparticles in order to to suppress nonspecific interactions in complex matrices. Antibodies with biotinylated PEG (polyethylene glycol) linkers are coupled to streptavidin-coated nanoparticles. Thereafter a second layer of linkers is added in order to surround the antibodies by a shell of linkers [4].

In a different approach we employ double-stranded DNA strands as rod-like spacers between the particle and the biomolecule. The DNA strands are labelled with biotin on one end, allowing them to bind directly to the streptavidin on the particle surface. The commercial availability of a large number of functional groups on DNA allows us to conjugate virtually any biomolecule including fluorescent dyes, antibodies, proteins, and enzymes.